1. Field of the Invention
The present invention relates to recombinant polypeptides that are useful for diagnosing American trypanosomiasis, or Chagas disease. Chagas disease is caused by the infectious agent Trypanosoma cruzi. More particularly, the invention relates to specific combinations of recombinant T. cruzi polypeptides, synthesized using genetic engineering techniques, and to constructs and processes for producing the recombinant polypeptides, and to an assay and kit for detecting T. cruzi infection which employs the recombinant polypeptides.
2. Background
Chagas disease is a zoonosis caused by the protozoan parasite, Trypanosoma cruzi. This organism is primarily transmitted through contact with its triatomine insect vectors, but transmission by transfusion of contaminated blood and congenital transmission also are important. Historically Chagas disease has been a public health problem in all of Latin America, with the exception of the Caribbean nations. The World Health Organization estimates that 16-18 million persons are chronically infected with T. cruzi, and that 45,000 deaths occur each year due to the illness. Infection with T. cruzi is life-long and specific drug treatment lacks efficacy and often causes serious side effects. Ten to thirty percent of T. cruzi-infected persons develop chronic symptomatic Chagas disease, and the burden of disability and mortality in the endemic countries is enormous.
An estimated 80,000 to 100,000 T. cruzi-infected persons now live in the United States. These immigrants pose a risk for transfusion-associated transmission of the parasite here and in other countries to which Latin Americans have emigrated. Eight such cases have been reported in the United States, Canada, and Europe, all of which occurred in immunosuppressed patients in whom acute T. cruzi infection was diagnosed because of the fulminant course of the illness. Most transfusions are given to immunocompetent patients in whom acute Chagas disease would be a mild illness, and thus it is reasonable to assume that many other undetected instances of transfusion-associated transmission of T. cruzi have occurred in the United States and other industrialized nations. The question of whether blood donated in the United States should be screened serologically for antibodies to T. cruzi has been considered for at least a decade by both public and private entities involved in blood banking. A panel of experts convened in early 2000 by the American Red Cross to consider this issue recommended unanimously that our blood supply be screened serologically. Implementation of such a recommendation, however, is not an option currently because no test for T. cruzi infection has been cleared by the FDA for screening donated blood.
Diagnosis of T. cruzi infection presents problems. Demographic and clinical data are suggestive at best. Parasitologic tests, e.g., xenodianosis, hemoculture and PCR are insensitive. Other serologic tests are generally insensitive and lack specificity, as false positive reactions often occur with specimens from patients having infectious diseases, such as leishmaniasis, syphilis, or malaria; autoimmune diseases; and other parasitic and non-parasitic illnesses.
Such conventional tests include indirect immunofluorescence (IIF), indirect hemagglutination (IHA), and complement fixation (CF) tests, as well as enzyme-linked immunosorbent assays (ELISA or EIA). Due to the lack of sensitivity and specify of the three commonly used assays, when a sample has a positive result from any, the blood must be discarded. Table I shows that in a major Brazilian blood bank (Hemocentro, são Paulo, Brazil), up to 3.43% of blood donations fall into this category.
TABLE IIIFIHACF% w/ Results+++0.68%+−+0.71%++−−+++−−2.04%−+−−−+TOTAL:3.43%
Commercially available ELISAs include lysate-based tests such as the Chagas Enzyme Immunoassay (EIA), available from Abbott Laboratories of Abbott Park, Ill. (the subject of FDA 510(k) Premarket Notification No. K933716, herein incorporated by reference in its entirety); the Chagas' IgG ELISA, available from Meridian Bioscience, Inc. of Cincinnati, Ohio, and its predecessor, Gull Laboratories (the subject of FDA 510(k) Premarket Notification No. K911233, herein incorporated by reference in its entirety); and the Chagas' kit (EIA method), available from Hemagen Diagnostics, Inc., of Waltham, Mass. (the subject of FDA 510(k) Premarket Notification No. K930272, herein incorporated by reference in its entirely). However, because these tests have less than optimal sensitivities and specificities, their use for screening donated blood would fail to detect some T. cruzi-infected units and also would cause substantial numbers of otherwise usable units to be discarded needlessly.
One of the present inventors has previously developed a radioimmune precipitation assay (RIPA), described in Kirchhoff L V, Gam A A, Gusmao R D, Goldsmith R S, Rezende J M, Rassi A. “Increased specificity of serodiagnosis of Chagas' disease by detection of antibody to the 72 and 90 kDa glycoproteins of Trypanosoma cruzi.” J Infect Dis 1987; 155:561-564, herein incorporated by reference in its entirety. This test is considered the benchmark against which other tests are measured, and it is the only current option for confirmatory testing in the United States. Unfortunately, the RIPA costs $175 per assay, and at that price, screening the approximately 13 million units of blood donated each year would cost over $2 billion.
Therefore, the present inventors have further developed recombinant assays for detection of T. cruzi infection. A typical recombinant polypeptide and method for assaying is described by them in U.S. Pat. No. 5,876,734, No. 6,228,601, and PCT Publication No. WO 95/25797, each of which is herein incorporated by reference in its entirety. Such assays for T. cruzi infection based on recombinant antigens, in contrast to those utilizing native antigens (e.g., the conventional lysate-based assays), as discussed above, will be more accurate, i.e., the sensitivity and specificity will be higher.
Furthermore, the recombinant assays of the invention present manufacturing advantages over the materials for the RIPA and conventional tests. Once the molecular biology has been completed, the recombinant antigens are produced in Escherichia coli, thus eliminating completely any biohazard associated with growing the parasites in liquid culture. This is a substantive advantage, as many cases of laboratory-acquired T. cruzi infection have been reported. Additionally, recombinant antigens produced in E. coli are much easier to purify, quantitate, and standardize than antigen lysates produced in liquid cultures of parasites, thus facilitating the manufacture of a consistent product and simplifying compliance with governmental regulations. A final advantage lies in the fact that several of the recombinant proteins presented in this application are comprised of two to four distinct protein segments derived from separate T. cruzi genes. This use of hybrid recombinant proteins also facilitates manufacture of an assay in that several antigenically distinct proteins are obtained in a single purification, quantitation, and standardization run.